Molecular oxygen is an essential nutrient for nonfacultative aerobic organisms, including, of course, humans. Oxygen is used in many important ways, namely, as the terminal electronic acceptor in oxidative phosphorylation, in many dioxygenase reactions, including the synthesis of prostaglandins and of vitamin A from carotenoids, in a host of hydroxylase reactions, including the formation and modification of steroid hormones, and in both the activation and the inactivation of xenobiotics, including carcinogens. The extensive P-450 system uses molecular oxygen in a host of important cellular reactions. In a similar vein, nature employs free radicals in a large variety of enzymic reactions.
Excessive concentrations of various forms of reactive oxygen species and of free radicals can have serious adverse effects on living systems, including the peroxidation of membrane lipids, the hydroxylation of nucleic acid bases, and the oxidation of sulfhydryl groups and of other sensitive moieties in proteins. If uncontrolled, mutations and/or cellular death result.
Biological antioxidants include well-defined enzymes, such as superoxide dismutase, catalase, selenium glutathione peroxidase, and phospholipid hydroperoxide glutathione peroxidase. Nonenzymatic biological antioxidants include tocopherols and tocotrienols, carotenoids, quinones, bilirubin, ascorbic acid, uric acid, and metal-binding proteins. Various antioxidants, being both lipid and water soluble, are found in all parts of cells and tissues, although each specific antioxidant often shows a characteristic distribution pattern. The so-called ovothiols, which are mercaptohistidine derivatives, also decompose peroxides nonenzymatically.
Free radicals, particularly free radicals derived from molecular oxygen, are believed to play a fundamental role in a wide variety of biological phenomena. In fact, it has been suggested that much of what is considered critical illness may involve oxygen radical (“oxyradical”) pathophysiology (Zimmerman, J. J. (1991) Chest 100:189S). Oxyradical injury has been implicated in the pathogenesis of pulmonary oxygen toxicity, adult respiratory distress syndrome (ARDS), bronchopulmonary dysplasia, sepsis syndrome, and a variety of ischemia-reperfusion syndromes, including myocardial infarction, stroke, cardiopulmonary bypass, organ transplantation, necrotizing enterocolitis, acute renal tubular necrosis, and other disease. Oxyradicals can react with proteins, nucleic acids, lipids, and other biological macromolecules producing damage to cells and tissues, particularly in the critically ill patient.
Free radicals are atoms, ions, or molecules that contain an unpaired electron (Pryor, W. A. (1976) Free Radicals in Biol. 1:1). Free radicals are usually unstable and exhibit short half-lives. Elemental oxygen is highly electronegative and readily accepts single electron transfers from cytochromes and other reduced cellular components; a portion of the O2 consumed by cells engaged in aerobic respiration is univalently reduced to superoxide radical (i.e., .O2−) (Cadenas, E. (1989) Ann. Rev. Biochem. 58:79). Sequential univalent reduction of .O2− produces hydrogen peroxide (ie., H2O2), a hydroxyl radical (i.e., .OH), and water.
Free radicals can originate from many sources, including aerobic respiration, cytochrome P-450-catalyzed monooxygenation reactions of drugs and xenobiotics (e.g., trichloromethyl radicals, i.e., CCl3., formed from oxidation of carbon tetrachloride), and ionizing radiation. For example, when tissues are exposed to gamma radiation, most of the energy deposited in the cells is absorbed by water and results in scission of the oxygen-hydrogen covalent bonds in water, leaving a single electron on hydrogen and one on oxygen, thereby creating two radicals, i.e., H. and .OH. The hydroxyl radical, i.e., .OH, is the most reactive radical known in chemistry. It reacts with biomolecules, sets off chain reactions and can interact with the purine or pyrimidine bases of nucleic acids. Indeed, radiation-induced carcinogenesis may be initiated by free radical damage (Breimer, L. H. (1988) Brit. J Cancer 57:6). In addition, the “oxidative burst” of activated neutrophils produces abundant superoxide radical, which is believed to be an essential factor in producing the cytotoxic effect of activated neutrophils. Reperfusion of ischemic tissues also produces large concentrations of oxyradicals, typically superoxide (Gutteridge and Halliwell (1990) Arch. Biochem. Biophys. 283:223). Moreover, superoxide can be produced physiologically by endothelial cells for reaction with nitric oxide, a physiological regulator, forming peroxynitrite, i.e., ONOO− which may decay and give rise to hydroxyl radical, .OH (Marletta, M. A. (1989) Trends Biochem. Sci. 14:488; Moncada, et al. (1989) Biochem. Pharmacol 38:1709; Saran, et al. (1990) Free Rad. Res. Commun. 10:221; Beckman, et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87:1620). Additional sources of oxyradicals are “leakage” of electrons from disrupted mitochondrial or endoplasmic reticular electron transport chains, prostaglandin synthesis, oxidation of catecholamines, and platelet activation.
Oxygen, though essential for aerobic metabolism, can be converted to poisonous metabolites, such as the superoxide anion and hydrogen peroxide, collectively known as reactive oxygen species (ROS). Increased ROS formation under pathological conditions is believed to cause cellular damage through the action of these highly reactive molecules on proteins, lipids, and DNA. During inflammation, ROS are generated by activated phagocytic leukocytes. As described above; during the neutrophil “respiratory burst,” superoxide anion is generated by the membrane-bound NADPH oxidase. ROS are also believed to accumulate when tissues are subjected to ischemia followed by reperfusion.
Many free radical reactions are highly damaging to cellular components, i.e., they crosslink proteins, mutagenize DNA, and peroxidize lipids. Once formed, free radicals can interact to produce other free radicals and non-radical oxidants such as singlet oxygen (1O2) and peroxides. Degradation of some of the products of free radical reactions can also generate potentially damaging chemical species. For example, malondialdehyde is a reaction product of peroxidized lipids that reacts with virtually any amine-containing molecule. Oxygen free radicals also cause oxidative modification of proteins (Stadtman, E. R. (1992) Science 257:1220).
Aerobic cells generally contain a number of defenses against the deleterious effects of oxyradicals and their reaction products. Superoxide dismutases (SODs) catalyze the reaction:2.O2−+2H+- - ->O2H2O2which removes superoxide and forms hydrogen peroxide. H2O2 is not a radical, but it is toxic to cells and it is removed by the enzymatic activities of catalase and glutathione peroxidase (GSH-Px). Catalase catalyzes the reaction:2 H2O2- - ->2 H2O+O2and GSH-Px removes hydrogen peroxide by using it to oxidize reduced glutathione (GSH) into oxidized glutathione (GSSG) according to the following reaction:2 GSH+H2O2- - ->GSSG+2 H2OOther enzymes, such as phospholipid hydroperoxide glutathione peroxidase (PLOOH-GSH-Px), converts reactive phospholipid hydroperoxides, free fatty acid hydroperoxides, and cholesterol hydroperoxides to corresponding harmless fatty acid alcohols. Glutathione S-transferases also participate in detoxifying organic peroxides. In the absence of these enzymes and in presence of transition metals, such as iron or copper, superoxide and hydrogen peroxide can participate in the following reactions which generate the extremely reactive hydroxyl radical, i.e., .OH−:.O2−+Fe3+- - ->O2+Fe2+H2O2+Fe2+- - ->.OH+OH−+Fe3+
In addition to enzymatic detoxification of free radicals and oxidant species, a variety of low molecular weight antioxidants, such as glutathione, ascorbate, tocopherol, ubiquinone, bilirubin, and uric acid, serve as naturally-occurring physiological antioxidants (Krinsky, N. I. (1992) Proc. Soc. Exp. Biol. Med. 200:248–54). Carotenoids are another class of small molecule antioxidants and have been implicated as protective agents against oxidative stress and chronic diseases. Canfield, et al., (1992) Proc. Soc. Exp. Biol. Med. 200:260, summarize reported relationships between carotenoids and various chronic diseases, including coronary heart disease, cataract, and cancer. Carotenoids dramatically reduce the incidence of certain premalignant conditions, such as leukoplakia, in some patients.
In order to prevent the damaging effects of free radicals and free radical-associated diseases, great efforts have been made to develop new antioxidants that are efficient at removing dangerous oxyradicals, particularly superoxide and hydrogen peroxide, and that are inexpensive to manufacture, stable and possess advantageous pharmacokinetic properties, such as the ability to cross the blood-brain barrier and penetrate tissues. Most recently, Malfroy-Camine, et aL have achieved this goal with their unexpected discovery that members of a class of compounds that were originally described as epoxidation catalysts, the so-called salen-metal complexes, also exhibit potent superoxide dismutase activity and/or catalase activity and, thus, function effectively as catalysts for free radical removal both in vitro and in vivo (see, U.S. Pat. Nos. 5,403,834, 5,834,509, 5,696,109 and 5,827,880, all of which issued to Malfroy-Camine, the teachings of which are incorporated herein by reference). Prior to this discovery, the salen-transition metal complexes had only been described and used as chiral epoxidation catalysts for various synthetic chemistry applications (see, Fu, et al. (1991) J. Org. Chem. 56:6497; Zhang, W. and Jacobsen, E. N. (1991) J. Org. Chem. 56:2296; Jacobsen, et al. (1991) J. Am. Chem. Soc. 113:6703; Zhang et al. (1990) J. Am. Chem. Soc. 112:2801; Lee, N. H. and Jacobsen, E. N. (1991) Tetrahedron Lett. 32:6533; Jacobsen, et al. (1991) J. Am. Chem. Soc. 113:7063; Lee, et al. (1991) Tetrahedron Lett. 32:5055).
Malfroy-Camine, et al. have now found that salen-metal complexes are also useful as potent antioxidants for various biological applications, including their use as pharmaceuticals for the prevention and/or treatment of free radical-associated diseases. Pharmaceutical formulations, dietary supplements, improved cell and organ culture media, improved cryopreservation media, topical ointments, and chemoprotective and radioprotective compositions can now be prepared with an effective amount or concentration of at least one antioxidant salen-metal complex. In addition, Malfroy-Camine, et al. have found that salen-metal complexes can also be used to partially or totally arrest the progression of neurodegenerative diseases. For instance, antioxidant salen-metal complexes can be used for the treatment and prophylaxis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, etc. Other uses for such salen-metal complexes are disclosed in U.S. Pat. Nos. 5,403,834, 5,834,509, 5,696,109 and 5,827,880.
Although the contributions of Malfroy-Camine, et al. have revolutionized the field of antioxidants that are useful in the prevention and treatment of free radical-associated diseases, it would still be advantageous if salen-metal compounds having increased stability could be developed. The present invention fulfills this and other goals.